Screening methods for the identification of anti-HIV compounds employing a cyclosporine-specific monoclonal antibody that cross-reacts with HIV-1 p24

ABSTRACT

The subject invention provides new uses for novel derivatives of cyclosporine A and antibodies directed thereto. Specifically, the derivatives and antibodies are useful in methods of treating AIDS, methods of purifying the Gag protein of HIV, methods of screening for anti-HIV compounds, and methods for detecting HIV in a subject. The derivatives and antibodies can also be incorporated into a kit useful for screening for anti-HIV compounds.

This application is a continuation of U.S. Ser. No. 08/390,133, filedFeb. 17, 1995, now U.S. Pat. No. 5,604,092.

This invention was made with government support under Grant Numbers RO1NS-15581, RO1 H1-36581 and PO1 HL-36581 and training grants2-T32-AI-07161-11 and T32-CA-09503 from the National Institute ofHealth, U.S. Department of Health and Human Resources. Accordingly, theU.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byarabic numerals within parentheses. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the invention described and claimed inthis application.

Cyclosporine A (CsA) is a cyclic undecapeptide of fun-gal origin whichis a immunosuppressive agent useful in preventing organ rejection intransplant patients (1-3).

Because the therapeutic index of CsA is narrow, it is important tomeasure serum cyclosporine levels in patients treated with CsA (4). Thiscan be accomplished by high performance liquid chromatography or by RIA,with the latter procedure being the more convenient one.

It has reported, and we have confirmed (unpublished), that CSA, itself,is non-immunogenic (5). To obtain antibodies, therefore, it is necessaryto link CsA to a protein carrier. The side chains of CsA, however,consist only of aliphatic groups with none of the functional groupscustomarily used to link a hapten to a carrier. Previous workers havemade immunogenic cyclosporine C (CsC)-protein conjugates because the CsChas a threonine residue in position 2 (5). Linkage to a protein was viaa hemisucciniate, using a water soluble carbodiimide as a couplingagent. Polyclonal antisera were successfully raised in this way and areroutinely used to measure CsA in patients sera (5). More recently,monoclonal antibodies were prepared using an activated ester of alysyl-CsA derivative (6).

We have chosen to use CsA, itself, as a hapten by converting it to areactive carboxyl-containing peptide via a photochemical reaction.Coupling of this derivative to proteins has led to the successfulraising of CsA-specific rabbit antibodies that can be used to measureCsA levels in sera of transplant patients under treatment with CsA.

Recently, Luban et al. (30) have shown that HIV-1 Gag protein binds toCyclophilin A and B and that the Gag portion of SIV binds only toCyclophilin B. We therefore determined that since Gag and CsA bind tocyclophillin, an antibody directed against CsA might bind to Gag andthereby treat HIV infection by inhibition of HIV replication. Further itis believed that the compositions of matter made according to thisspecification would be useful to treat AIDS.

SUMMARY OF THE INVENTION

The present invention provides a molecule having the structure:

where each R may independently be H or X, provided that at least one Ris X, where X is a ligand which is produced as the result of aphotochemical reaction between a precursor of X containing aphotochemically activatable group and a hydrogen of cyclosporine A andwhich comprises a reactive group.

The invention further provides that the reactive group may be a groupwhich is reactive with a macromolecule. In a preferred embodiment ofthis invention, the macro-molecule may be a polypeptide. In a verypreferred embodiment, the invention further provides that the.polypeptide may be a protein. In a preferred embodiment, the reactivegroup may be a carboxyl.

Specific examples of X may include but are not limited to the following:

In a preferred embodiment of the invention, the probability is greaterthat 0.75 that only one R in the aforementioned molecule is X. In a verypreferred embodiment, the probability is about 1.0.

The present invention further provides a molecule which comprises acongener of cyclosporine A characterized by the structural backbone ofcyclosporine A in which one or more hydrogen atoms are replaced by oneor more ligands, each such ligand both comprising a reactive group andbeing attached to the structural backbone of cyclosporine A at alocation which a hydrogen atom has been replaced as the result of aphotochemical reaction between a precursor of the ligand containing aphotochemically activatable group and the hydrogen atom being replaced.

The present invention further provides an immunosuppressive agent usefulfor preventing organ rejection in a transplant subject comprising anamount of the aforementioned molecules effective to inhibit organrejection in a transplant subject and a pharmaceutically acceptablecarrier.

The present invention also provides a composition of matter whichcomprises a conjugate of a compound and the aforementioned moleculewherein the compound is bound to the molecule through the reactive groupof the ligand X.

The invention further provides a composition of matter which comprises aconjugate of a macromolecule and the aforementioned molecule wherein themacromolecule is bound to the molecule through the reactive group of theligand X.

Similarly, the invention provides a composition of matter whichcomprises a conjugate of a polypeptide and the aforementioned moleculewherein the polypeptide is bound to the molecule through the reactivegroup of the ligand X.

Moreover, the invention provides a composition of matter which comprisesa conjugate of a protein and the aforementioned molecule wherein theprotein is bound to the molecule through the reactive group of theligand X.

The invention also provides a method for preventing rejection in atransplant subject comprising administering to the subject an amount ofthe aforementioned molecule effective to inhibit organ rejection in thetransplant subject.

The subject invention further provides an antibody directed to theaforementioned composition of matter specific for cyclosporine A orcongener of cyclosporine A. In accordance with the teachings of theinvention the antibody may further be characterized as polyclonal ormonoclonal. In addition, these antibodies may be detectably labeled.

The invention further provides a method of detecting the presence ofcyclosporine A or congener of cyclosporine A in a biological tissuesample which comprises treating the biological tissue sample with theaforementioned detectably labeled antibody under conditions permittingthe antibody to bind to cyclosporine A or congener and form a complextherewith, removing labeled antibody which is not bound to cyclosporineA or congener, detecting the presence of labeled antibody bound tocyclosporine A or congener and thereby detecting the presence ofcyclosporine A or congener in the biological tissue sample.

The invention further provides another method of detecting the presenceof cyclosporine A or a congener of cyclosporine A in a biological tissuesample which comprises treating the biological tissue sample with theaforementioned unlabeled antibody under conditions permitting theantibody to bind to cyclosporine A or congener and form a complextherewith, removing antibody which is not bound to cyclosporine A orcongener, treating the complex with a labeled antibody directed to theunlabeled antibody under conditions such that the labeled antibody bindsto the unlabeled antibody of the complex, removing labeled antibodywhich is not bound to the complex, detecting the presence of labeledantibody bound to the complex and thereby detecting the presence ofcyclosporine A or congener in the biological tissue sample.

Additionally, this invention provides a method of determining theconcentration of cyclosporine A or congener of cyclosporine A in abiological fluid sample which comprises, contacting a solid support withan excess of the aforementioned composition of matter under conditionspermitting the composition of matter to attach to the surface of thesolid support, contracting a predetermined volume of biological fluidsample with a predetermined amount of the aforementioned labeledantibody under conditions such that the cyclosporine A or congener inthe sample binds to the labeled antibody and forms a complex therewith,contacting the resulting complex to the solid support to the surface ofwhich the composition of matter is attached under conditions permittingthe labeled antibody of the complex to bind to the composition ofmatter, treating the solid support so that only the composition ofmatter and labeled antibody of the complex bound thereto remain,quantitatively determining the amount of labeled antibody of the complexbound to the composition of matter, and thereby determining theconcentration of cyclosporine A or congener in the biological fluidsample.

This invention provides another method of determining the concentrationof cyclosporine A or congener of cyclosporine A in a biological fluidsample which comprises contacting a solid support with an excess of theaforementioned composition of matter under conditions permitting thecomposition of matter to attach to the solid support, contacting apredetermined volume of biological fluid sample with a predeterminedamount of the aforementioned antibody under conditions such that thecyclosporine A or congener in the sample binds to the antibody and formsa complex therewith, contacting this complex with a predetermined amountof labeled antibody directed to the unlabeled antibody under conditionssuch that the labeled antibody binds to the unlabeled antibody complexof the prior step and forms a labeled complex therewith, contacting theresulting labeled complex to the solid support to the surface of whichthe composition of matter is attached under conditions permitting theunlabeled antibody bound to the labeled antibody of the labeled complexto bind to the composition of matter, treating the solid support so thatonly the composition of matter and labeled complex bound thereto remain,quantitatively determining the amount of labeled antibody of the labeledcomplex bound to the unlabeled antibody which is in turn bound to thecomposition of matter, and thereby determining the concentration ofcyclosporine A or congener in the biological fluid sample.

The invention also provides a method of determining the concentration ofcyclosporine A or congener of cyclosporine A in a biological fluidsample by radioimmunoassay which comprises radioactively labeling apredetermined amount of a substance comprising cyclosporine A, congenerof cyclosporine A or the aforementioned composition of matter, addingthe predetermined amount of radiolabeled substance to the biologicalfluid sample, contacting this mixture with a predetermined amount of theaforementioned unlabeled antibody under conditions suitable to permitthe antibody to bind to the cyclosporine A or congener in the biologicalfluid sample and the labeled substance, removing any unboundradiolabeled substance, quantitatively determining the amount of labeledsubstance bound to the antibody, and thereby determining theconcentration of cyclosporine A or congener in the biological fluidsample.

The invention also provides a method of monitoring levels ofcyclosporine A or congener of cyclosporine A in a subject whichcomprises taking biological fluid samples from a subject atpredetermined intervals and determining the amount of cyclosporine A orcongener in each biological fluid sample according to the aforementionedassays.

The invention additionally provides a method for producing a monoclonalauto-anti-idiotypic antibody which comprises contacting lymphoid callsof an animal under suitable conditions with an effectiveantibody-raising amount of the aforementioned composition of matter,collecting the lymphoid cells at a suitable time after the contacting,fusing the collected lymphoid cells with appropriate myelona cells toproduce a series of hybridoma cells each of which produces a monoclonalantibody, screening under suitable conditions the series of hybridomacells so produced to identify those which secrete a monoclonal antibodycapable of binding to an antibody directed to the aforementionedcomposition of matter, separately culturing a hybridoma cell soidentified in an appropriate medium, and separately recovering undersuitable conditions the monoclonal anti-idiotypic antibody produced bythe hybridona cell.

The invention further provides an antibody directed to theaforementioned monoclonal auto-anti-idiotypic antibody. Additionally,the invention provides an antibody directed to the aforementionedantibodies. These antibodies directed to other antibodies may be used inan immunoregulatory substance useful for preventing organ rejection in atransplant subject in an amount effective to inhibit organ rejection ina transplant subject and a pharmaceutically acceptable carrier.

The invention further provides a method of reducing the amount ofcyclosporine A or congener in a subject which comprises administeringintravenously to the subject an amount of the aforementioned antibodyeffective to reduce the amount of cyclosporine A and permitting theantibody to bind to the excess cyclosporine A, thereby rendering theexcess cyclosporine A ineffective.

The invention also provides a method of reducing the amount ofendogenous immunoregulatory substances, or other biologically activesubstances which are endogenous, which share epitopes with cyclosporineA or congener of cyclosporine A in a subject which comprisesadministering intravenously to the subject an amount of aforementionedantibody or fragment thereof effective to reduce the amount ofendogenous substances and permitting the antibody or fragment thereof tobind to the excess endogenous substances, thereby rendering the excessendogenous substances ineffective.

The invention further provides a method of testing the potential of apharmacological agent as an immunoactive agent which comprises runningan immunochemical assay competitive between the pharmacological agentand known amounts of labeled cyclosporine A or congener of cyclosporineA with the aforementioned antibody under conditions such that theantibody forms complexes with the pharmacological agent and cyclosporineA or congener and determining the displacement from the antibody oflabeled cyclosporine A or congener by the pharmacological agent.

In addition, the present invention provides a composition of matterwhich comprises aminodextran and the aforementioned molecule, whereinthe aminodextran is bound to the molecule through the reactive group ofligand X.

The invention also provides a method of testing a pharmalogical agentfor immunosuppressive activity which comprises contacting cells with thecomposition of matter above under conditions such that the compositionof matter causes agglutination of cells, contacting the resultingagglutinated cells with the pharmalogical agent, an inhibition ofagglutination being indicative that the pharmalogical agent hasimmunosuppressive activity.

The subject invention also provides a method of treating AIDS in asubject which comprises administration of an antibody which specificallybinds to N-methyl leucine residues 9 and 10 of cyclosporine to a subjectin an amount effective to inhibit HIV replication and thereby treatAIDS.

The subject invention further provides a method of treating AIDS in asubject which comprises administration of a composition of matter,wherein the composition of matter comprises a compound having thestructure:

wherein a plurality of R's are X and the remainder are H, wherein X is aligand comprising a reactive group and wherein X is bonded to thecompound by a photochemical reaction between a hydrogen of cyclosporineA and a photochemically activatable precursor of X;

and a polypeptide coupled to the compound through the reactive group onX;

in an amount effective to produce an antibody which specifically bindsto N-methyl leucine residues 9 and 10 of cyclosporine to inhibit HIVreplication and thereby treat AIDS.

The subject invention also provides a method of purifying the Gagprotein of HIV which comprises:

a) contacting a sample known to contain the Gag protein with an antibodywhich specifically binds to N-methyl leucine residues 9 and 10 ofcyclosporine under conditions such that the antibody binds to and formsa complex with the Gag protein;

b) isolating the antibody-protein complex formed in (a); and

c) separating the protein from the isolated antibody-protein complexfrom (b).

The subject invention also provides a method of screening for anti-HIVcompounds which comprises:

a) immobilizing a composition of matter having the structure:

wherein a plurality of R's are X and the remainder are H, wherein X is aligand comprising a reactive group and wherein X is bonded to theCompound by a photochemical reaction between a hydrogen of cyclosporineA and a photochemically activatable precursor of X;

b) contacting the immobilized composition of matter from (a) with amixture of the compound suspected of having anti-HIV activity and adetectably labeled antibody which specifically binds to N-methyl leucineresidues 9 and 10 of cyclosporine under conditions ailowing for thelabeled antibody to bind to the immobilized composition of matter from(a) and form a complex therewith;

c) separating any unbound labeled antibody from the complex formed in(b);

d) detecting any labeled antibody bound to the complex in (c); and

e) quantitating the amount of labeled antibody from (d).

The subject invention further provides a kit for of screening foranti-HIV compounds which comprises:

a) a plate comprising a plurality of wells;

b) a composition of matter immobilized upon the wells, wherein thecomposition of matter has the structure:

wherein a plurality of R's are X and the remainder are H, wherein X is aligand comprising a reactive group and wherein X is bonded to thecompound by a photochemical reaction between a hydrogen of cyclosporineA and a photochemically activatable precursor of X; and

c) a solution comprising a detectably labeled antibody whichspecifically binds to N-methyl leucine residues 9 and 10 ofcyclosporine.

The subject invention also provides a method for detecting HIV in asubject which comprises:

a) obtaining a serum sample from a subject;

b) contacting the serum sample with a detectably labeled antibody whichspecifically binds to N-methyl loucine residues 9 and 10 of cyclosporineunder conditions permitting the detectably labeled antibody to bind toand form a complex with a Gag protein on any HIV in the serum sample;

c) removing any antibodies which are not part of the complex of (b); and

d) detecting the presence of antibodies in the serum sample, therebydetecting the presence of HIV in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Photochemical reaction between CsA and BBA.

FIG. 2. Scatchard plot of binding data.

FIG. 3. Inhibition of the binding CsA to R575 by various cyclosporinederivatives.

FIG. 4. Titers (ng/ml) of patients' sera as determined by RIA using R575and Sandoz antibody.

FIGS. 5A and 5B. FIG. 5A is a structural diagram of cyclosporine A(CsA). FIG. 5B is a three-dimensional space-filling model of CsA basedon X-ray crystallography and NMR data.

FIGS. 6A-6E. ELISA inhibition curves for one representative of each ofthe specificity groups defined in the Experimental Details Section.Conditions of the assay are also described therein. The CsA derivativesused in this study are as follows: CsA (◯), CsA-BBa (⋄), CsC (▪), 582(□), 665 (▴), 243 (X), 032 (•), 039 (Δ), 717 (+).

FIGS. 7A-7C. Specific recognition of two opposite regions of the Csmolecule exposed on D-Lys⁸-Cs-BSA and Thr²-Cs-BSA conjugates by 22 McAbsbelonging to the 5 groups of specificity. The D-Lys⁸-Cs-BSA (□) andThr²-Cs-BSA (□) conjugates were coated on the solid phase at aconcentration of 0.5 μg/ml. Negative control for coating is shown (▪).The supernatants of McAb-producing hybridomas were used at dilutionscorresponding to the beginning of the plateau of maximum ELISA reactionwith the best recognized Cs-BSA conjugate.

FIGS. 8A and 8B. ELISA inhibition curves of the reaction of McAb B-111.4 with D-Lys⁸-Cs-BSA (FIG. 8 A) and Thr²-Cs-BSA (FIG. 8B) conjugatescoated on the solid phase (0.25 μg/ml). The following Cs-derivativeswere used as inhibitors: CsA (◯), 3′-acetyl-MeBmt-Ce (x), Thr²-Cs (▪),MeAla⁶-Cs (Δ) and MeIle¹¹-Cs (•).

FIG. 9. Western Blot in which Gag of HIV-1 was gel electrophoresed,transferred to a nitrocellulose membrane and cut into strips. Each stripwas tested for binding to our various monoclonal anti-CsA antibodies antto an antibody raised by immunization with Gag (column l). Column 9represents action of B-11 1.4. Multiple bands are the results of partionproteolysis of Gag.

FIG. 10. ELISA in which plate was coated with P24 and competition isseen between cyclophilin A and B-11 1.4.

FIG. 11. ELISA in which P24 is used to coat plate and competition isseen between B-11 1.4 and CsA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a molecule having the structure:

wherein each R may independently be H or X, provided that at least one Ris X, where X is a ligand which is produced as the result of aphotochemical reaction between a precursor of X containing aphotochemically activatable group and a hydrogen of cyclosporine A andwhich comprises a reactive group.

The invention further provides that the reactive group may be a groupwhich is reactive with a macromolecule. Examples of such macromoleculesinclude, but are not limited to, polysaccharides, complex carbohydrates,and any organic polymers including but not limited to polyacrylamide,polynitrocellulose, and polystyrene. In a preferred embodiment of thisinvention, the macro-molecule may be a polypeptide. In a very preferredembodiment, the invention further provides that the polypeptide may be aprotein.

In a further embodiment of the invention, the reactive group may be anester, carbonyl, amine or phosphonamide. In a preferred embodiment, thereactive group may be a carboxyl.

Photochemical reactions are well-known in the art (7) and it is to beunderstood that X may be any ligand which is produced as the result of aphotochemical reaction between a precursor of X containing aphoto-chemically activatable group and a hydrogen of cyclosporine A andwhich comprises a reactive group.

Specific examples of X may include but are not limited to the following:

In a preferred embodiment of the invention, the probability is greaterthan 0.75 that only one R in the aforementioned molecule is X. In a verypreferred embodiment, the probability is about 1.0.

The present invention further provides a molecule which comprises acongener of cyclosporine A in which one or more hydrogen atoms arereplaced by one or more ligands, each such ligand both comprising areactive group and being attached to the structural backbone ofcyclosporine A at a location which a hydrogen atom has been replaced asthe result of a photochemical reaction between a precursor of the ligandcontaining a photochemically activatable group and the hydrogen atombeing replaced.

Congeners of cyclosporine A currently exist in the literature (5, 8) andit is anticipated that many more may be developed. It is foreseen thatthe novelties of the subject application which are applicable tocyclosporine A may also be applicable to such congeners.

The basic structure of cyclosporine A is represented in FIG. 5A.Examples of such congeners include, but are not limited to, cyclosporineA with:

(a) alanine at position 2;

(b) threonine at position 2;

(c) valine at position 2;

(d) norvaline at position 2 and 5; and

(e) alpha-amino butyric acid at position 7.

The present invention further provides an immunosuppressive agent usefulfor preventing organ rejection in a transplant subject comprising anamount of the aforementioned molecules effective to inhibit organrejection in a transplant subject and a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers. Such carriersare well-known in the art and may include, but are not intended to belimited to, any of the standard pharmaceutical carriers such asphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents.

The aforementioned immunosuppressive compositions may be superior tocyclosporine A in several ways. First, the compositions may avoid thetoxicity problems inherent with cyclosporine A, specifically kidneydamage. Second, these compositions may be soluble and thereby preferablefor dosage regulation.

The present invention also provides a composition of matter whichcomprises a conjugate of a compound and the aforementioned moleculewherein the compound is bound to the molecule through the reactive groupof the ligand X. The general process for preparation of antigenichapten-carrier conjugates is known in the art (9).

The invention further provides a composition of matter which comprises aconjugate of a macromolecule and the aforementioned molecule wherein themacromolecule is bound to the molecule through the reactive group of theligand X.

Similarly, the invention provides a composition of matter whichcomprises a conjugate of a polypeptide and the aforementioned moleculewherein the polypeptide is bound to the molecule through the reactivegroup of the ligand X.

Moreover, the invention provides a composition of matter which comprisesa conjugate of a protein and the aforementioned molecule wherein theprotein is bound to the molecule through the reactive-group of theligand X. Again, it is to be understood that the scope of the inventionincludes any protein capable of being bound to the molecule. Specificexamples of this protein includes bovine serum albumin, rabbit serumalbumin, keyhole limpet hemocyanin, ovalbumin, or any globulin includingbut not limited to thyroglobulin.

The invention also provides a method for preventing rejection in atransplant subject comprising administering to the subject an amount ofthe aforementioned molecule effective to inhibit organ rejection in thetransplant subject.

The subject invention further provides an antibody directed to theaforementioned composition of matter specific for cyclosporine A orcongener of cyclosporine A. In accordance with the teachings of theinvention, the antibody, as cited herein and in the following usesthereof, may further be characterized as polyclonal or monoclonal.

In addition, these antibodies may be detectably labeled. Such labels arewell-known in the art and include but are not limited to enzyme labels,fluorescent labels, and radioactive labels such as fluorophore orbiotinylated labels.

The invention further provides a method of detecting the presence ofcyclosporine A or congener of cyclosporine A in a biological tissuesample which comprises treating the biological tissue sample with theaforementioned detectably labeled antibody under conditions permittingthe antibody to bind to cyclosporine A or congener and form a complextherewith, removing labeled antibody which is not bound to cyclosporineA or congener, detecting the presence of labeled antibody bound tocyclosporine A or congener and thereby detecting the presence ofcyclosporine A or congener in the biological tissue sample.

Detecting the presence of cyclosporine A or congener in biologicaltissue sample is useful since the toxic effects of cyclosporine Ainclude damage to tissues, particularly kidney. Accordingly, in apreferred embodiment of the method of detecting the presence ofcyclosporine A or congener, the biological tissue sample is kidney.However, the biological tissue sample is not intended to be limited tokidney and includes other biological tissues such as liver.

Additionally, this invention provides a method of determining theconcentration of cyclosporine A or congener of cyclosporine A in abiological fluid sample which comprises, contacting a solid support withan excess of the aforementioned composition of matter under conditionspermitting the composition of matter to attach to the surface of thesolid support, contacting a predetermined volume of biological fluidsample with a predetermined amount of aforementioned labeled antibodyunder conditions such that the cyclosporine A or congener in the samplebinds to the labeled antibody and forms a complex therewith, contactingthe resulting complex to the solid support to the surface of which thecomposition of matter is attached under conditions permitting thelabeled antibody of the complex to bind to the composition of matter,treating the solid support so that only the composition of matter andlabeled antibody of the complex bound thereto remain, quantitativelydetermining the amount of labeled antibody of the complex bound to thecomposition of matter, and thereby determining the concentration ofcyclosporine A or congener in the biological fluid sample.

The aforementioned biological fluid and the biological fluid used in thefollowing methods for determining the concentration of cyclosporine A orcongener thereof and method for monitoring levels of cyclosporine A orcongener thereof, may be, but is not limited to blood, urine, feces orextracts of tissue.

This invention provides another method of determining the concentrationof cyclosporine A or congener of cyclosporine A in a biological fluidsample which comprises contacting a solid support with an excess of theaforementioned composition of matter under conditions permitting thecomposition of matter to attach to the surface of the solid support,contacting a predetermined volume of biological fluid sample with apredetermined amount of the aforementioned antibody under conditionssuch that the cyclosporine A or congener in the sample binds to theantibody and forms a complex therewith, contacting this complex with apredetermined amount of labeled antibody directed to the unlabeledantibody under conditions such that the labeled antibody binds to theunlabeled antibody complex of the prior step and forms a labeled complextherewith, contacting the resulting labeled complex to the solid supportto the surface of which the composition of matter is attached underconditions permitting the unlabeled antibody bound to the labeledantibody of the labeled complex to bind to the composition of matter,treating the solid support so that only the composition of matter andlabeled complex bound thereto remain, quantitatively determining theamount of labeled antibody of the labeled complex bound to the unlabeledantibody which is in turn bound to the composition of matter, andthereby determining the concentration of cyclosporine A or congener inthe biological fluid sample.

In the two aforementioned methods of determining the concentration ofcyclosporine A or congener, the composition of matter may be attached tothe surface of the solid support by covalent or noncovalent bonds.

The invention also provides a method of determining the concentration ofcyclosporine A or congener of cyclosporine A in a biological fluidsample by radioimmunoassay which comprises radioactively labeling apredetermining amount of a substance comprising cyclosporine A, congenerof cyclosporine A or the aforementioned composition of matter, addingthe predetermined amount of radiolabeled substance to the biologicalfluid sample, contacting this mixture with a predetermined amount of theaforementioned unlabeled antibody under conditions suitable to permitthe antibody to bind to the cyclosporine A or congener in the biologicalfluid sample and the labeled substance, removing any unboundradiolabeled substance, quantitatively determining the amount of labeledsubstance bound to the antibody, and thereby determining theconcentration of cyclosporine A or congener in the biological fluidsample.

Methods of determining the concentration of cyclosporine A or congenerin the biological fluid sample from data concerning labeled complex iswell-known in the art. One such example includes comparing the data to astandard curve.

It is to be understood that it is within the scope of the presentinvention to use other types of assays and detectable labels with theaforementioned antibodies for determining the concentration ofcyclosporine A in a biological fluid sample.

The invention also provides a method of monitoring levels ofcyclosporine A or congener of cyclosporine A in a subject whichcomprises taking biological fluid samples from a subject atpredetermined intervals and determining the amount of cyclosporine A orcongener in each biological fluid sample according to the aforementionedassays.

The invention additionally provides a method for producing a monoclonalauto-anti-idiotypic antibody which comprises contacting lymphoid cellsof an animal under suitable conditions with an effectiveantibody-raising amount of the aforementioned composition of matter,collecting the lymphoid cells at a suitable time after the contacting,fusing the collected lymphoid cells with appropriate myeloma cells toproduce a series of hybridoma cells each of which produces a monoclonalantibody, screening under suitable conditions the series of hybridomacells so produced to identify those which secrete a monoclonal antibodycapable of binding to an antibody directed to the aforementionedcomposition of matter, separately culturing a hybridoma cell soidentified in an appropriate medium, and separately recovering undersuitable conditions the monoclonal anti-idiotypic antibody produced bythe hybridoma cell. Methods of producing monoclonal auto-anti-idiotypicantibodies are previously known in the art as outlined in U.S. Pat. No.5,114,010 issued Sep. 1, 1992, the contents of which are herebyincorporated by reference.

The invention further provides an antibody directed to theaforementioned monoclonal auto-anti-idiotypic antibody. Additionally,the invention provides an antibody directed to each of theaforementioned antibodies which are specific for cyclosporine A orcongener thereof. These antibodies may be used- in an immunoregulatorysubstance useful for preventing organ rejection in a transplant subjectin an amount effective to inhibit organ rejection in a transplantsubject and a pharmaceutically acceptable carrier.

The invention further provides a method of reducing the amount ofcyclosporine A or congener in a subject which comprises administeringintravenously to the subject an amount of the aforementioned antibodyeffective to reduce the amount of cyclosporine A and permitting theantibody to bind to the excess cyclosporine A, thereby rendering theexcess cyclosporine A ineffective.

The invention also provides a method of reducing the amount ofendogenous immunoregulatory substances, or other biologically activesubstances which are endogenous, which share epitopes with cyclosporineA or congener of cyclosporine A in a subject which comprisesadministering intravenously to the subject an amount of aforementionedantibody or fragment thereof effective to reduce the amount ofendogenous substances and permitting the antibody or fragment thereof tobind to the excess endogenous substances, thereby rendering the excessendogenous substances ineffective.

The invention also provides a method of testing a pharmalogical agentfor immunosuppressive activity which comprises contacting cells with thecomposition of matter above under conditions such that the compositionof matter causes agglutination of cells, contacting the resultingagglutinated cells with the pharmalogical agent, an inhibition ofagglutination being indicative that the pharmalogical agent hasimmunosuppressive activity. Preferably, the cells above are either T orB-cells.

The subject invention also provides a method of treating AIDS in asubject which comprises administration of an antibody which specificallybinds to N-methyl leucine residues 9 and 10 of cyclosporine to a subjectin an amount effective to inhibit HIV replication and thereby treatAIDS.

In a preferred embodiment of the above described invention the antibodywhich specifically binds to N-methyl leucine residues 9 and 10 ofcyclosporine is the monoclonal antibody B-11 1.4 produced by thehybridoma cell line having ATCC Accession No. HP-11853 HP-11853. Thishybridoma was deposited on Feb. 17, 1995 in accordance with the BudapestTreaty with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md., 20852 U.S.A.

The monoclonal antibodies of the subject invention include wholemonoclonal antibodies as well as antigen binding fragments thereof.Examples of such fragments are well known to those of ordinary skill inthe art and are referred to as Fab, Fab' or F(ab')2 antibody fragments.In a preferred embodiment the monoclonal antibody fragment is a Fab'fragment. In another-preferred embodiment the monoclonal antibodyfragment is a F(ab')₂ fragment. Methods of producing the antibodyfragments are also known to those of ordinary skill in the art. By wayof example, the Fab' fragment of the above-disclosed monoclonal antibodycan be produced by papain digestion of the monoclonal antibody.Similarly, the F(ab')₂ fragment can be produced by pepsin digestion ofthe monoclonal antibody.

In one embodiment of this invention the monoclonal antibody is a murineantibody. In another embodiment the monoclonal antibody is a chimericmonoclonal antibody. In still another embodiment the monoclonal antibodyis a humanized monoclonal antibody. However, in the preferred embodimentthe monoclonal antibody is a human monoclonal antibody.

For the purposes of this invention, a “chimeric” monoclonal antibody isa murine monoclonal antibody comprising constant region fragments(F_(c)) from a different animal. In a preferred embodiment of thisinvention, the chimeric monoclonal antibody comprises human FC andmurine F_(ab). For the purposes of this invention, a “humanized”monoclonal antibody is a murine monoclonal antibody in which humanprotein sequences have been substituted for all the murine proteinsequences except for the murine complement determining regions (CDR) ofboth the light and heavy chains.

In an additional embodiment the subject antibody, or fragment thereof,can be linked to a cytotoxic agent to form an immunotoxin. Toxins usefulas cytotoxic agents coupled to antibodies are well known to those ofordinary skill in the art and include, but are not limited to, toxinsfrom bacteria diphtheria, pseudomonas or shigella or the castor beantoxin ricin. Examples of the use of toxins coupled to antibodies or toprotein fragments for expression in cells to treat HIV infection includeHarrison et al., U.S. Pat. No. 5,306,631, issued Apr. 26, 1994 andArdman, U.S. Pat. No. 5,252,556, issued Oct. 12, 1993, the contents ofwhich are hereby incorporated in their entirety into the subjectspecification by reference.

The subject invention further provides a method of treating AIDS in asubject which comprises administration of a composition of matter,wherein the composition- of matter comprises a compound having thestructure:

wherein a plurality of R's are X and the remainder are H, wherein X is aligand comprising a reactive group and wherein X is bonded to thecompound by a photochemical reaction between a hydrogen of cyclosporineA and a photochemically activatable precursor of X:

and a polypeptide coupled to the compound through the reactive group onX;

in an amount effective to produce an antibody which specifically bindsto N-methyl leucine residues 9 and 10 of cyclosporine to inhibit HIVreplication and thereby treat AIDS.

In the practice of the above-described method the polypeptide is aprotein. Proteins useful in the practice of the claimed invention areknown to those of ordinary skill in the art and include but are notlimited to bovine serum albumin, rabbit serum albumin, and keyholelimpit heomycin.

In the practice of the methods of treatment of this invention thecompositions of matter may be administered as part of a pharmaceuticalcomposition which comprises any of the compositions of matter orantibodies and a pharmaceutically acceptable carrier. In the preferredembodiment of this invention, the compounds are administered to thesubject as a pharmaceutical composition.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers such as anorganic or inorganic inert carrier material suitable for enteral orparenteral administration which include, but are not limited to, water,gelatin, gum arabic, lactose, starches, magnesium stearate, talc,vegetable oils, polyalkylene glycols, petroleum gelly, etc. Thepharmacological preparations can be made up in solid form such astablets, dragees, suppositories or capsules, or in liquid form such assolutions, suspensions, or emulsions. The preparations may be sterilizedand/or contain adjuvants such as preserving, stabilizing, wetting oremulsifying agents, salts for varying the osmotic pressure, or buffers.Such preparations may also contain other therapeutic agents.

For the purposes of this invention, the term “pharmaceutically effectiveamount” of the compound means any amount of the compound which, whenincorporated in the pharmaceutical composition, will be effective toinhibit HIV replication and thereby treat AIDS but less than an amountwhich would be toxic to the subject. In the practice of this inventionthe amount of the composition of matter incorporated in thepharmaceutical composition may vary widely. Factors considered whendetermining the precise amount are well known to those skilled in theart. Examples of such factors include, but are not limited to, thesubject being treated, the specific pharmaceutical carrier and route ofadministration being employed and the frequency with which thecomposition is to be administered. In a preferred embodiment of thisinvention, the pharmaceutically effective amount of the compound is inthe range of 10 picomolar to 10 millimolar. In a particularly preferredembodiment the pharmaceutically effective amount is about 10 micromolar.

In the practice of this invention, the administration of the compositionmay be effected by any of the well known methods including, but notlimited to, oral, intravenous, intraperitoneal, intramuscular,subcutaneous or topical administration.

The subject invention also provides a method of purifying the Gagprotein of HIV which comprises:

a) contacting a sample known to contain the Gag protein with an antibodywhich specifically binds to N-methyl leucine residues 9 and 10 ofcyclosporine under conditions such that the antibody binds to and formsa complex with the Gag protein;

b) isolating the antibody-protein complex formed in (a); and

c) separating the protein from the isolated antibody-protein complexfrom (b).

In a preferred embodiment of the above-described method step (a) furthercomprises immobilizing the antibody on a matrix and contacting theprotein with the immobilized antibody. In the practice of this methodthe matrix may be any matrix known to those of ordinary skill in the artwhich allows for the immobilization of an antibody. Examples includeagarose beads, PVC microtiter plates and nitrocellulose, either as asheet or as fused to a microtiter plate. In a preferred embodiment thematrix comprises agarose beads.

An example of the practice of one embodiment of the invention whenactivated beads are used comprises (1) dialyzing the antibody against ofbinding buffer of 0.5 M sodium phosphate (pH 7.5); (2) preparing asolution of the antibody at the desired concentration in 0.5 M (pH 7.5)at a ratio of between 5 to 10 mg of antibody per milliliter of beads toyield a high-capacity column; (3) adding the activated beads, preparedby any method known to those of skill in the art; and (4) mixingovernight at approximately 4° C. with constant agitation.

In another preferred embodiment of the above-described method step (a)further comprises:

i) contacting the antibody with biotin under conditions such that theantibody is biotinylated;

ii) contacting the solution known to contain the Gag protein with thebiotinylated antibody from step (i) to form a complex with the Gagprotein; and

iii) contacting the biotinylated antibody-protein complex from (ii) witha matrix comprising streptavidin and agarose under conditions such thatthe biotin binds to the streptavidin in the matrix.

As an example of the practice of the above-described method thebiotinylated antibodies are prepared by (1) combining a biotin ester,for example comprising a solution of N-hydroxysuccinimide biotin at 10mg/ml in dimethyl sulfoxide, with the antibody at a ratio of between25-250 μg of ester per milligram of antibody incubating at roomtemperature for approximately 4 hr; (2) adding approximately 20 μl of 1MNH₄Cl per 250 μg of ester and incubate for approximately 10 min at roomtemperature; (3) adding sufficient biotinylated antibody to the solutioncontaining the Gag protein to bind the majority of the antigen(determined by prior titrations); (4) incubating at approximately 4° C.for approximately 1 hour; (5) at a temperature of approximately 4° C.,passing the antigen-antibody solution through a volume of uncoupledagarose beads equivalent to or larger than the volume of thestreptavidin-agarose beads that will be used to collect theantibody-antigen complex; and (6) at a temperature of approximately 4°C., collecting the biotinylated antibodies by passing theantigen-antibody solution through a streptavidin-agarose column at arate of approximately 7.5 to 10 ml/hr;

An example of the condition of the separation of step (c) above includes(1) washing the column with an appropriate number of volumes of bindingbuffer; and.(2) eluting the antigen by sequentially passing samples ofelution buffer through the column and collecting the fractions eluted.

In a preferred embodiment of any of the above-described methods theantibody which specifically binds to N-methyl leucine residues 9 and 10of cyclosporine is the monoclonal antibody B-11 1.4 produced by thehybridoma cell line having ATCC Accession No. HB-11853.

The subject invention also provides a method of screening for anti-HIVcompounds which comprises:

a) immobilizing a composition of matter having the structure:

 wherein a plurality of R's are X and the remainder are H, wherein X isa ligand comprising a reactive group and wherein X is bonded to thecompound by a photochemical reaction between a hydrogen of cyclosporineA and a photochemically activatable precursor of X;

b) contacting the immobilized composition of matter from (a) with amixture of the compound suspected of having anti-HIV activity and adetectably labeled antibody which specifically binds to N-methyl leucineresidues 9 and 10 of cyclosporine under conditions allowing for thelabeled antibody to bind to the immobilized composition of matter from(a) and form a complex therewith;

c) separating any unbound labeled antibody from the complex formed in(b);

d) detecting any labeled antibody bound to the complex in (c); and

e) quantitating the amount of labeled antibody from (d).

As an example of the practice of the above-described method (1) thecomposition of matter is bound to the bottom of wells, preferably PVCmicrotiter plates, by adding approximately 50 μl of a solution of thecomposition of matter (approximately 20 μg/ml), all dilutions done inPBS; (2) incubating the plates at room temperature for approximately 2hours; (3) washing the plates at least twice with PBS; (4) filling theplates with a blocking buffer, for example 3% BSA/PBS with 0.02% sodiumazide incubating at room temperature for between approximately 2 hoursto overnight; (5) washing the plates at least twice with PBS; (6) addingthe mixture of the labeled antibody and compound suspected of havinganti-HIV activity and a detectably labeled antibody, incubating at roomtemperature for approximately 2 hours; and (7) removing unboundantibodies and compound by multiple washes with PBS.

In a preferred embodiment of the above-described method the detectablylabeled antibody which specifically binds to N-methyl leucine residues 9and 10 of cyclosporine is the monoclonal antibody B-11 1.4 produced bythe hybridoma cell line having ATCC Accession No. HB-11853.

In the practice of the above-described method the detectably labeledantibody is labeled with an enzyme, dye, fluorescent marker, coloredbead, radioactive isotope or biotin.

In a preferred embodiment the compound suspected of having anti-HIVactivity will be a polypeptide.

The subject invention further provides a kit for of screening foranti-HIV compounds which comprises:

a) a plate comprising a plurality of wells;

b) a composition of matter immobilized upon the wells, wherein thecomposition of matter has the structure:

 wherein a plurality of R's are X and the remainder are H, wherein X isa ligand comprising a reactive group and wherein X is bonded to thecompound by a photochemical reaction between a hydrogen of cyclosporineA and a photochemically activatable precursor of X; and

c) a solution comprising a detectably labeled antibody whichspecifically binds to N-methyl leucine residues 9 and 10 ofcyclosporine.

In a preferred embodiment of the above described kit the detectablylabeled antibody which specifically binds to N-methyl leucine residues 9and 10 of cyclosporine is the monoclonal antibody B-11 1.4 produced bythe hybridoma cell line having ATCC Accession No. HB-11853.

In the use of the above-described kit the detectably labeled antibody islabeled with an enzyme, dye, fluorescent marker, colored bead,radioactive isotope or biotin.

In a preferred embodiment the wells will comprise PVC plates.

The subject invention also provides a method for detecting HIV in asubject which comprises:

a) obtaining a serum sample from a subject;

b) contacting the serum sample with a detectably labeled antibody whichspecifically binds to N-methyl leucine residues 9 and 10 of cyclosporineunder conditions permitting the detectably labeled antibody to bind toand form a complex with a Gag protein on any HIV in the serum sample;

c) removing any antibodies which are not part of the complex of (b); and

d) detecting the presence of antibodies in the serum sample, therebydetecting the presence of HIV in the subject.

In the practice of the above-described method the antibody may beimmobilized such as being bound to agarose beads or nitrocellulose orPVC as described above.

In a preferred embodiment of the above-described method the detectablylabeled antibody which specifically binds to N-methyl leucine residues 9and 10 of cyclosporine is the monoclonal antibody B-11 1.4 produced bythe hybridoma cell line having ATCC Accession No. HB-11853.

In the practice of the above-described method the detectably labeledantibody is labeled with an enzyme, dye, fluorescent marker, coloredbead, radioactive isotope or biotin.

Practice of, and determination of the optimal conditions for, any of themethods of the subject invention is within the skill of the ordinaryartisan. A standard laboratory text useful in the practice of theimmunohistochemical assays of the invention is Harlow and Lane,“Antibodies: A Laboratory Manual” (Cold Spring Harbor Laboratory, NewYork: 1988), the contents of which are hereby incorporated into thepresent specification by reference thereto.

This invention is illustrated in the Experimental Details section whichfollows. This section is set forth to aid in an understanding of theinvention but is not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS EXAMPLE I

A. Materials and Methods

4-Benzyoylbenzoic acid (eBa) was purchased from Aldrich Chemicals.Bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA),and N-hydroxysuccinimide were from Sigma Chemical.Dicyclohexylcarbodiimide was from Fluka. Cyclosporin A (CsA), [³H]CsA(SOCi/mMole), Cyclosporin “RIA-kits” and the various modifiedderivatives were generous gifts from Sandoz Ltd., Basel, Switzerland.[³H]CsA (17Ci/mMole) was purchased from Amersham. Kieselgel (silica gel60 F254) was purchased from E. Merck (cat. no. 5766).

B. Photolysis reaction

CsA (104 mg, 83 μmoles) was mixed with 36 mg (160 μmoles) of BBa in 0.6ml of benzene. The solution was purged with nitrogen gas and photolyzedat 320nm with a Spectroline B100 UV lamp (Spectronics, Westbury, L. I.)for 7 hours at a distance of 8 cm, at room temperature. Approximately 1microcurie of [³H] dihydro CsA was added as a tracer prior to exposureto UV. After photolysis, the benzene was evaporated in a rotating stillin vacuo and the dried produce dissolved in 1.5 ml of methanol. Theproduct was isolated by preparative thin layer chromatography on silicagel, in a solvent system of CHCl₃/methanol (85/15). Two major bands weeseen: Rf=0.58 and 0.72. The slower moving band (i.e. the product of thereaction, CsA-BBa) was eluted with methanol, and counted forradioactivity.

C. Hapten-Protein Conjugate

CsA-BBa (5.5 mg, ca. 4 μmoles) was added to 1 ml solution containing 552μg (4.8 μmoles) of N-hydroxysuccinimide (NHS) and 825 μg (4 Amoles) ofdicyclohexylcarbodiimide in 1 ml of methanol. The reaction was allowedto run overnight at room temperature and ester formation was detectedwith a neutral Fe-hydroxanate test (10-11).

Carrier proteins (BSA, RSA, or OVA) (10 mg; 0.14 μmole) were dissolvedin 1.0 ml of distilled H₂O, and the pH adjusted to 9.0 with M Na₂CO₃.CsA-BBa-NHS (5.2mg; 3.6 μmoles) in 1.0 ml of methanol was added dropwiseto the protein solution. After all was added, the pH was readjusted to 9and the reaction allowed to proceed overnight at room temperature. Thereaction mixture was then dialyzed against PBS for 24 hours and countedfor radioactivity to determine coupling efficiency. About 6-7cyclosporins were coupled to each molecule of BSA, RSA, or OVA. Theconjugates were further purified by gel filtration HPLC (LKB TSK 3000).Confirmation of the linkage of CsA to the proteins came from RIAinhibition experiments. Quantitation is not possible by this techniquebecause there was no way to determine the valence of the conjugate as acompetitive inhibitor, i.e., how many of the haptens linked to theprotein took part in the inhibition reaction.

D. Other CyA-BBa Derivatives

The following amide derivatives of CyA-BBa, ethanolamide,monoamino-hexanediamine, amide of D-Lysine methyl ester, D-glucamide,and stearylamine were produced using the same methods above and themethods of example III by reacting ethanolamine, hexanediamine,D-lysine-O-methyl ester, and octadecialamine, respectively, withN-hydroxysuccinimide ester of CyA-BBa.

E. Immunization

Two female New Zealand White rabbits were immunized intradermally alongwith the back, with a 1:1 (v:v) mixture of CsA-BBa-BSA in completeFreund's adjuvant (1 mg/ml of antigen). The rabbits were boosted withCsA-BBa-BSA in incomplete Freund's adjuvant at 3-4 week intervals andbled weekly following each boost. Both rabbits responded by producingcyclosporine-specific antibodies. The sera of one rabbit, R575, wascharacterized further.

F. Radioimmunoassay

Serum antibodies were detected by a modification of the publishedradioimmunoassay (5, 12). Serum (100 μl) diluted in Sandoz buffer A (50mM Tris, pH 8.5) was added to 200 μl of [³H]CsA in Sandoz buffer B (50mM Tris, pH 8.5; 0.1% Tween 20) containing 2% horse serum, and incubatedfor 2 hours at room temperature or overnight at 4° C. Binding by dilutedpreimmune serum was used as a control. Free and bound ligand wereseparated with charcoal supplied by Sandoz according to their procedure.

G. Determination of antibody specificity

Antibody specificity was determined by an inhibition RIA, using a panelof size CsA analogues, modified at different amino acid positions. Thecyclosporin derivatives were dissolved in 100% ethanol at aconcentration of 5.0 mg/ml, stored at −20° C., and diluted to finalconcentrations of 0.27 nM to 2.7 μM in Sandoz buffer B for theinhibition experiments. A constant dilution of rabbit antibody, inbuffer A, was added to 200 μl of buffer B containing [³H] dihydro CsAand different amounts of inhibitor, and incubated overnight at 4° C.Inhibition curves for each CsA derivatives were generated.

H. Detection of CsA in sera of transplant patients

Cyclosporin levels in the sera of 25 transplant patients were determinedby an inhibition RIA, using either our rabbit anticyclosporin antibodiesdiluted 1:600 or a polyclonal antibody preparation supplied by Sandoz,as part of their kit. Diluted rabbit anticyclosporin antiserum (100 μl)or Sandoz polyclonal antibody were added to 100 μl [3H]CsA in buffer Band 100 μl of patient's serum prediluted either 1:5 (for Sandozantibody) or 1:15 (for our rabbit antibody) in buffer B, containing 2%horse serum. Sera from three different patients, taken before they hadbegun cyclosporin treatment, were used as controls. Samples wereincubated overnight at 4° C., and CsA levels were calculated bycomparing the level of inhibition to a standard curve obtained withknown amounts of cyclosporin.

I. Scatchard Analysis

The binding constant of the rabbit antibodies was determined byScatchard analysis. Different concentrations of [³H] dihydro CsA,ranging from 10 nM to 0.1 nM, were added to a constant amount ofantibody and allowed to incubate overnight at 4° C., bound ligand wasdetermined by the RIA described above.

Results

CsA lacks chemically active groups that can be used for conjugation toproteins. Therefore, a novel procedure was developed for the purpose ofintroducing carboxyl groups into the molecule. This procedure,photochemical in nature, inserts a carboxyl-containing molecule (BBa)into the alkyl side chains of CSA (FIG. 1), presumably but notcertainly, at random.

Antibodies generated in rabbits with the CsA-BBa-BSA conjugate wereexamined for specificity and affinity by RIA. Scatchard analysis (FIG.2) revealed a relatively homogenous population of high affinityantibodies, with Kd−9.8±2.8×10⁻¹¹M.

The specificity of the antibodies for various cyclosporin derivativeswas determined by an inhibition RIA. The results are shown in FIG. 3 andTable I. The derivatives can be divided roughly into three groupsaccording to their affinities: CsA, CsD, 665, 243 and 032 are in thehigh affinity group. CsC, 582 and 039 are of moderate affinities; 717inhibits very poorly.

Shown in Table II are the results of assays of cyclosporin levels in thesera of patients undergoing CsA treatment subsequent to cardiactransplantation. Titers were determined using our antibodies and thepolyclonal antibodies in the Sandoz kit. Also tabulated in Table II aredata supplied by the laboratory of the Department of Surgery. Asillustrated in FIG. 4, in our hands the levels determined with ourantibody agreed with results using the commercial kit. Linear regressionof analysis of the data yields a slope of 0.88 and a correlationcoefficient of 0.84.

Discussion

The α, β unsaturated ketone, BBa, is among reagents that, uponphotoactivation by U.V. light, can insert into aliphatic side chains(7). It was selected for this study because its photoactive intermediatedoes not cleave peptide bonds (13). This is an important considerationbecause it has been shown that a single break in a peptide bond of CsA,such as in iso-CsA, which has lost a peptide bond by an N O shift, leadsto loss of activity even though, in the case of Iso-CsA, a cyclicstructure is maintained. Apparently an altered conformation leads to abiologically inactive molecule.

The insertion of BBa into CsA is probably a somewhat random process,although we have not attempted to characterize the various products. Ifrandom, we are generating populations of antibodies that recognizedifferent residues of the CsA molecule. We have tried to learn somethingabout these antibodies by doing inhibition studies with a panel ofcyclosporin derivatives (FIG. 3, Table I). First of all, the relativelyshallow slopes of the curves indicate that the immune response is oligoor polyclonal, probably the former. If it were monoclonal, 90%inhibition would occur at a tenfold higher concentration than 10%inhibition. A second important observation is that 100% inhibition of[³H] CsA binding can be obtained with all of the competing cyclosporinderivatives except 717, which, however, is certainly capable of morethan 50% inhibition. These results indicate that all of the cyclosporinderivatives compete for the total population of antibodies specific forCsA.

The inhibition data in Table I and FIG. 3 indicate that the variouscyclosporine derivatives can be divided into three groups with respectto their affinities for the population of antibodies in the immune sera.CsA, CsD, 665, 243 and 032 bind best. Moderate affinities are shown byCsC, 582 and 039. The results with 717 indicate low affinity. Derivative717 differs from CsA by having a bulky O-t-butyl-D-serine of D-alanineat position 8. This could implicate position 8 as a dominant epitope. Onthe other hand, introduction of a bulky group at position 8, in place ofthe compact methyl group of D-alanine, could distort the cyclosporinemolecule markedly (6).

The derivatives showing moderate affinities, CSC, 582 and 039, aresubstituted at position 2, 3 and 6 respectively.

TABLE 1 IC₅₀ ^(a) of Various Analogues of CsA Derivative^(b) IC₅₀ (nM)CsD 4.3 665 4.8 A 6.0 243 6.0 032 7.0 CsC 11.5 582 12.0 039 18.0 7172700 ^(a)IC₅₀ = Concentration for 50% inhibition ^(b)The derivativeslisted differ from CsA in the following ways: CsD, valine replacesα-aminobutyric acid at position 2; 665, 0-acetylthreonine replacesα-aminobutyric acid at position 2; 243, hydroxyl group of(4R)-4-[(E)-2-butenyl]-4-N-dimethyl-L-threonine in position 1 isacetylated; 032, N-methyl-isoleucine replaces N-methylvaline at position11; CsC, threonine replaces α-aminobutyric acid at position 2; 582,proline replaces sarcosine at position 3; 039, N-methyl-D-alanineN-methylleucine at position 6; 717, 0-t-butyl-D-serine replacesD-alanine at position 8.

TABLE 2 Cyclosporine Titers in Patients' Sera (ng/ml) Hospital Patient #R575^(a) Commercial^(b) Laboratory^(c)  1  51 undetectable  30  2 190170 128  3 195 180 156  4 135 120  76  5 175 180 180  6 135 110  88  7195 195 164  8 205 185 172  9 240 225 245 10 210 210 215 11 113 125 18012  95 100 124 13  77 113 124 14  98 105  88 15  84 100 112 16 124 163152 17 165 250 205 18 135 190 180 19 141 158 132 20 231 250 188 21 126153 134 22  81 113  88 23 117 145 110 24 107 175 148 25  57  60  71^(a)Antibodies prepared as described above. ^(b)Antibody from kit fromSandoz, Ltd. Assay run in our laboratory. ^(c)Results reported byhospital laboratory using Sandoz Kit.

None of the substitutions are bulky. However, the substitution ofproline for sarcosine at position 3 is known to disturb the conformationof 582 at positions 3 and 4 (14).

Those derivatives having affinities similar to that of CsA aresubstituted in positions 1, 2 and 11, all clustered at one “face” of thecyclic peptide. The substitutions, however, are not drastic with respectto size differences of the side chains. A definitive study of thespecificity of the antisera and correlation with conformation andbiological activity requires testing with a larger number of cyclosporinanalogues, which are available (14).

Assay of cyclosporin levels in patients' sera is feasible with thisantibody preparation. Our results (FIG. 4 and Table II) are in goodagreement with cyclosporin levels determined using commercial (Sandoz)antisera prepared by immunization with a protein conjugate of CsC. Themoderate discrepancies probably indicate differences in crossspecificities of the antibodies for metabolites of cyclosporine, (5),which is to be expected since our antigen differs from the antigen usedto produce the commercial antiserum.

EXAMPLE II

Preparation of CyA-BSA Conjugate and CyA-Sepharose Affinity Column

The character of the side chains of CyA (i.e., an absence of amino orcarboxyl groups) precluded the use of conventional coupling procedures,except possibly to the unusual “C-9-amino acid” in positionl(N-methyl-(4R)-4-butenyl-(L)-threonine) (15-17). However, modificationof this amino acid was ill advised since this residue was critical tothe biological activity of CyA. CyC has threonine instead ofτ-aminobutyric acid at the second amino position (AA2). This analog isbiologically active and has been used to prepare cyclosporine-proteinconjugates using the hemisuccinate derivative (15, 5). As noted byKahan, however, coupling to this residue was likely to lead to stearicinterference with the “active” portion of the molecule (18). Thisconclusion was based on substitution studies in which it had been shownthat amino acids 11, 1, 2, and 3 were critical for immunosuppressiveactivity (17). Because of this possibility, we used a photochemicalprocedure that has provided random links to the various exposed methylor methylene groups of CyA. By having populations of CyA derivativesheterogeneous with regard to attachment sites, it was insured that aportion of the molecules could be coupled to protein without the activeamino acids being buried.

We first reacted p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (19,20) with a large excess of aminohexanoic acid in anhydrous dimethylformamide. The reaction mixture was incubated in the dark at roomtemperature for 18 hours, following a similar procedure described bySamuels and coworker (21). The product was operated by preparative TLC(21) and has the following formula:

Next CyA, which is very soluble in all organic solvents except hexane(17), was mixed with the above product in a benzene solvent andphotolyzed. Two moles of carbene precursor to one mole cyclosporine Awere also used. The mixture was irradiated with UV light from a mercuryarc (mainly 254 nm). Reaction conditions were empirically chosen toavoid multiple substitutions of CyA molecules. Derivatized CyA (CyA-Hex)were separated by TLC. To provide a functional group that reacted withamino groups, the carboxyl group(s) of CyA-Hex were activated byconversion to the N-hydroxy succinimide (NHS) ester in the presence ofdicyclohexylcarbodiimide. Then, CyA-Hex-NHS was dissolved in anhydrousdimethyl formamide and added as a small volume to bovine albumin andkeyhole limpet hemocyanin in pH-8.8 bicarbonate buffer and incubatedovernight at 4° C. These conditions have been found to work well withother NHS derivatives (22, 23). To determine the degree of substitutionof protein amino acid groups by CyA-Hex, we used the trinitrobenzenesulfonic acid procedure of Habeeb (24). In our previous studies, ESA andKLH conjugates were both found to work well as immunogens and asantigens in solid phase immunoassays. All chemicals needed for thepreparation of these reagents were commercially available.

To prepare a CyA affinity column, an excess of CyA-Hex-NHS in anhydrousdimethyl formamide was reacted with aminohexyl-Sepharose 4B(AH-Sepharose 4B), suspended and swollen in the same solvent. Thismatrix, which was prepared by a carbonyldiimidazole coupling procedure,avoided the introduction of the ion exchange groups associated with thefrequently used cyanogen bromide coupling and reduced leakage due to thecross-linked agarose and the stable carbonyldiimidazole linkage (25).Trinitrobenzene sulfonic acid will be used to get a semiquantitativeestimate of residual amino groups on Sepharose beads.

EXAMPLE III

A. Production and Characterization of Nonoclonal Antibodies

The production and characterization of monoclonal antibodies specificfor Cyclosporine A has previously been reported in Cacalano et al., Mol.Immunol., Vol. 29, pages 107-118 (1991), the entire contents of whichare hereby incorporated by reference.

1. Reagents

Cyclosporine A (Cs, Sandimmune), and the various modified Csderivatives, including CsC, 665, 032, 243, 582, 039, and 717 wereobtained from Sandoz Ltd. Basel, Switzerland. The structural diagram andspace-filling model of Cs are shown in FIG. 5B. Structural informationfor the Cs derivatives is as follows: CsC, threonine replacesα-aminobutric acid at position 2: 582, proline replaces sarcosine atposition 2; 665. 0-acetyl threonine replaces α-aminobutyric acid atposition 2; 243, hydroxyl group of(4R)-4-[(E)-2-butenyl]-4-N-dimethyl-L-threonine at position 1 isacetylated; 032, N-methylisoleucine replaced N-methyl-valine at position11; 039, N-methyl-D-alanine replaces N-methyl isoleucine at position 6;717, 0-t-butyl-D-serine replaces D-alanine at position 8.

[³H]CsA (17 Ci/mmol) was purchased from Amersham Corp., (ArlingtonHeights, Ill.). 4-Benzoylbenzoic acid (BBa) was purchased from AldrichChemicals (Milwaukee, Wis.). Bovine serum albumin (BSA), ovalbumin(Ova), and polyethylene glycol (PEG) 8000 were from Sigma Chemical Corp.(St. Louis, Mo.). Bovine gamma globulin was purchased from Mann ResearchLaboratories, Inc. (New York, N.Y.). Dextran G10 was from Pharmacia LKBBiotechnology (Upsala, Sweden). Norit A (charcoal) was purchased fromFisher Scientific (Springfield, N.J.). Fetal calf serum (FCS) was fromHyclone (Logan, Utah). Peroxidase-conjugated goat anti-mouse IgG+IgM waspurchased from TAGO (Burlingame, Calif.). Purified human livercyclophilin (CyP) was a generous gift from Dr. Robert E. Handshumacher,Yale University. The monoclonal antibody isotyping kit was from Zymed(San Francisco, Calif.).

2. Hybridomas

The Cs photolysis reaction with BBA, purification of Cs-BBa, andconjugation of Cs-BBa to BSA and Ova have been described previously(Cacalano et al., 1989). BALB/c mice (Charles River) were immunized i.p.with approximately 0.10 mg of Cs-BBa-BSA emulsified in CFA. Mice wereboosted at three week intervals with 0.1 mg Cs-BBa-BSA emulsified inincomplete Freund's adjuvant (IFA), over a period of six months. Threedays before fusion, mice were injected i.v. with 50 μg of Cs-BBa-BSA inPBS. Spleen cells were fused with nonsecreting myeloma cellsP3×63-AgS.653 (Kearney et al., 1979), according to the method of Sharonet al. (1979). Two weeks later, the hybridoma supernatant was assayedfor the presence of anti-cyclosporine antibodies by ELISA (see below).The ELISA positive clones were confirmed for Cs binding inradioimmunoassay. Clones positive by RIA were subcloned twice bylimiting dilution. The class and subclass of each antibody weredetermined by ELISA.

3. ELISA for anti-Cyclosporine Monoclonal Antibody screening

Polystyrene microplates (Corning 25855) were coated with 500 ng/mlCs-BBa-Ova in 0.1 M sodium bicarbonate, pH 9.3, overnight at 4° C.Plates were washed with PBS containing 0.1% Tween 20 (PBS-Tween 20), an150 μl of culture supernatant were incubated in the wells for 1 hr at37° C. Plates were washed four times with PBS-Tween, and {fraction(1/3000)} dilution of horseradish peroxidase-labeled goat anti-mouseIgG+IgM in PBS-Tween was added to each well. After washing the platesfour times with PBS-Tween, 150 μl of substrate (7 mg o-phenylenediaminedihydrochloride in 10 ml of 0.1 M citrate-phosphate buffer, pH 4.8,containing 5 μl of 30% H₂O₂) was added. The reaction was stopped after10 mins by the addition of 50 μl 8 N H-SO₄, and the absorbance of eachwell was measured at 490 nm on a Dynatech Microplate reader.

A second ELISA Procedure used conjugates of D-Lys⁸-Cs-BSA andThr²-Cs-BSA for coating (Queeniaux et al., 1987b). An indirect detectionsystem consisting of rabbit anti-mouse Ig followed by alkalinephosphates-conjugated goat anti-rabbit IgG was performed as previouslydescribed (Queenlaux et al., 1987b).

4. Compatities of Monoclonal Antibodies with cyclophilin

Polystyrene microplates were coated with 500 ng/ml of Cs-BBA-Ova in 0.1M NaHCO₃, pH 9.3, overnight at 4C. Hybridoma supernatant was diluted in0.1 M Na-PO₄, pH 7.0. Cyclophilin was added to final concentration of1.6×10⁻⁷ M-1×10⁻¹¹ M, and incubated in the microtiter wells for 1 hr at37° C. Bound McAbs were detected as described above. The results wereexpressed as percentage of inhibition in the presence of CyP, relativeto the O.D. 490 nm measured in the absence of CyP.

5. Radioimmunoassay

For McAb screening, 100 μl of hybridoma supernatant was added to 100 μlof RIA buffer B (50 mM Tris, pH 6.5 containing 0.1% Tween 20) and 100 μlbuffer B containing [³H]Ca (final concn 1 nM) and incubated for 2 hr atroom temp or overnight at 4° C.

Bound was separated from free [³H]Cs by the addition of 150 μl SephadexC10-coated charcoal (Norit A) in RIA buffer A (50 mM Tris, pH 8.5),incubated for 12 minat 4° C., followed by centrifugation. Thesupernatant, containing bound [³H] Cs, was counted for radioactivity.

For Scatchard analyses of high affinity antibodies, 100 μl of dilutedhybridome supernatant was incubated with [³H]CsA at concns from1.0×10⁻⁵M to 5.0×10⁻¹¹ M, overnight at 4° C. Bound from free [³H]Cs wasseparated by incubation with dextran coated charcoal as described above.

For Scatchard analyses of low affinity antibodies, diluted hybridomasupernatant was incubated with [³H]Cast concs of 5'10⁻⁵ M-5×10⁻¹⁰M overnight at 4° C. Bound from free [³H]Cs was separated by the addition of50 μl of a 10 mg/ml solution of bovine gamma globulin in buffer A,followed by the addition of 200 μl of 30% PEC 8000 in buffer A,vertexing and incubating for 10 min at 4° C. Tubes were centrifuged for10 min and the pellets were redissolved in 500 μl distilled H₂O andcounted for radioactivity.

B. Results

Of the 2 McAbs generated, 1E are IgGl, K, B9 1.2 is IgG2b, k and A112.10, G2, 2.1, and B-1 1.4 are IgM, k.

The McAs were first characterized as to epitope specifically in anELISA, with Cs-BBA-ovalbumin as the coating antigen, Cs, Cs-BBa (theimmunizing hapten), or one of seven Cs analogs, singly substituted atdifferent amino acid residues was examined for inhibition.

A typical set of inhibition of curves is shown in FIGS. 6A-6E. Bycomparing the relative potancies of the inhibitors, antibody specificityfor epitopes on Cs were determined. The antibodies were studied fortheir sensitivity to the drastically altered IC₅₀ as compared toinhibition with Cs. As shown in FIGS. 6A-6B and Table 3, the antibodiescan be arranged into five groups, each with uniques bindingcharacteristics. Derivatives giving an IC₅₀<50-fold different fromunmodified Cs for all members of a group were considered to be modifiedat a position involved in the contact with the antibody combining site.

Group I contains antibodies that bind to derivative 665, which ismodified at position 2 (α-L-aminobutyric acid to O-actylthreonina), witha much higher affinity than they bind to Cs. They are also highlysensitive to the O-t-butyl-D-serine substitution at position 8 inderivative 717, which completely abolishes the inhibitory activity of717.

The antibodies in group II are sensitive to the substitutions in Csderivatives 243 (acetylation of hydroxy group of MeBmt at position 1).665 (O-acetyl threonine in place of α-aminobutyric acid at position 2),and 582 (sarocsine to proline substitution at position 3, which causes aconformational change at positions 3 and 4).

Antibodies in group III are sensitive to the substitutions in Csderivatives 243, 665, 582, 039, and 717.

Antibodies in group IV are sensitive to the substitution at position 8in derivatives 717 but are relatively insensitive to other side chainmodifications in the Cs derivatives used in the experiment.

Antibodies in group V recognize three derivatives with a much loweraffinity than Cs. In the case of derivative 582, the antibody issensitive to a conformational changes at position 3 and 4, induced bythe sarocsine to proline substitution at amino acid 3. They also poorlyrecognize derivative 039, which contains an N-methylisoleucine toN-methyl-D-alanine substitution at position 6, and are also highlysensitive to the D-Ala to O-t-butyl-D-serine substitution at position 8in derivative 717.

The binding of the 22 McAbs to D-Lys⁸-Cs-BSA and Thr³-Cs-BSA conjugates,in which opposite sides of the Cs molecules are likely to be exposed,was analyzed in direct ELISA. As shown in FIGS. 7A-7C, some McAbs couldbind selectively to one of the two conjugates, whereas other McAbs boundequally well to both conjugates. For example, all members of groups Iand V bound only the Thr²-Ca-BSA conjugate, the McAbs in group II boundonly to the D-Lys⁵-C-BSA conjugate, and Abs in groups III and IV boundboth conjugates group III and IV bound both conjugates.

Titrations of hybridoma supernatant on the two conjugates wereperformed, and the ratio of he McAB concentrations required to achievesimilar ELISA reaction with D-Lys⁶-Cs-BSA and Thr²-Cs-BSA is reported inTable 4. Direct binding of the McAbs to the different Cs conjugates wasin agreement with the initial grouping based on the ELISA inhibitionstudies.

The binding of the 22 McAbs to these two Cs-BSA conjugates was alsofound to correlate well with their fine specificity patterns, ascharacterized with a set of 28-36 Cs derivatives. Typical ELISA curvesshowing McAb binding either to D-Lys⁸-Cs-BSA (panel A) or to Thr²-Ca-BSA(panel B) and inhibited by Cs or Cs derivatives are shown in FIGS.8A-8B, D-Lys⁸-Cs-BSA was subsequently used for coating in the inhibitionexperiments, except for those McAbs binding selectively to Thr²-Cs-BSA(see Table 4 and FIGS. 7A-7C). An example of the binding of 26 Csderivatives by McAbs belonging to each of the five specificity groupsmentioned above is reported in Table 5. The results are expressed as thedifference of IC₅₀ (in log₁₀) between the Cs derivatives and Cs. AllCs-derivatives used in this study were selected for having a conservedpeptide ring conformation compared with CsA (as determined in NMR orX-ray diffraction).

McAb G71.1 belonging to group 1, was sensitive to substitutions of Csresides 3, 5, 8 an d9, whereas it did not discriminate between Cs and Csderivatives substituted at other amino acid positions.

McAb H41.3 (group II) was sensitive to modifications at residues 1-6,indicating that this McAb might recognize a more extended epitope.Within residue 1, this McAb could differentiate between modifications ofthe first carbon of the McBmt side chain (C3′) and modification of thelast carbon of this side chain (C8′). When C3′, hydroxylated in Cs, wasacetylated or dehydroxylated, the IC₅₀ for that derivative was >10-foldhigher than Cs. In contrast, H41.3 did not discriminate between Cs andCs-derivatives showing hydroxylation of C8′ or missing this last carbon(in Ethyl MeThr¹-Cs).

McAb H61.8 (group III) was mostly sensitive to modifications of residues2, 3, 4, and 6, and also weakly) recognized amino acid 1.

McAb B-11 1.4 (group IV) seemed to have a very different pattern ofrecognition since only modifications of residues 9 and 10 led to adecrease of binding >10-fold compared to Cs.

McAb C4 2.2 (group V) was sensitive to modifications of residues 2, 6,8, 9 and 10.

Similar studies were performed with all of the 22 McAbs, using 28-36Cs-derivatives. It was striking that McAbs from different specificitygroups exhibit very different recognition patterns. Within each group,however, the recognitives patterns of the McAbs for the different Csderivative was strikingly similar. One exception was McAB B9 1.2, whichbelongs to group III by the first two criteria mentioned above (initialELISA inhibition with a limited number of Cs derivatives, and binding ofboth the D-Lys⁸-Cs-BSA and Thr²-Ca-BSA conjugates), but showed a finespecificity very similar to that of group II McAbs; i.e., it wassensitive to modification of C3′ of residue 1, and substitutions ofresidues 2, 4, 5 and 6.

Based on these results, we can identify residues that are recognized bythe different McAbs, as summarized in Table 4, When some substitution ata given residue produced IC₅₀ values threshold (+) to greater thantenfold (++) higher than that observed with Cs, it was concluded thatthe particular residue was important for McAb binding. When allderivatives modified at a given position did not produce significantlydifferent IC₅₀ values (−), that amino acid was assumed not interact withthe McAb combining site.

TABLE 3 Relative IC₅₀ ^(a) values of cyclosporine derivatives formonoclonal antibodies. Antibody CsA CsC CsA-BBa 582 665 243 032 039 717GROUP I G22.1 1.0 1.0 0.1  0.25   0.007 ^(b)  9.0 2.4 9.0 >1000 B41.21.0 0.9 0.0014 0.4  0.0005 10.6 1.8 3.2 >1000 H12.4 1.0 1.2 0.05 1.30.004 21.7 2.4 11.2 >1000 A111.1 1.0 1.8 0.002 1.1 <0.001   61.0 7.043.7 >1000 H21.2 1.0 4.2 0.8 3.0 0.2  68.5 3.4 10.0 >1000 C91.0 1.0 1.31.3 3.6 0.01  20.0 17.0 20.0 >1000 D111.3 1.0 1.6 0.5 1.1 0.004 15.0 5.815.0 >1000 G71.1 1.0 5.6 0.75 5.0 0.02  13.0 11.0 16.0 >1000 GROUP IID31.3 1.0 25.0 0.007 >10000  3217     342  1.0 145 11.0 D51.4 1.0 50.01.0 >10000  10000      1600  2.5 115 115 H41.3 1.0 6.0 6.0 >1000 >300       117 2.4 4.0 8.0 GROUP III C42.2 1.0 3.2 0.2 >10000  5000    272  9.0 >10000 180 H61.5 1.0 59.0 ND >10000 1000     2650 59 >10000 315B91.2 1.0 2.0 11.0 >1000  >1000       >1000 5.4 1000 222 GROUP IVA112.10 1.0 0.8 0.2 3.0 134     84.2 2.7 1.0 200 D101.1 1.0 0.9 1.4 1.54.3  31.0 6.7 2.7 75.0 B22.4 1.0 5.8 2.1 3.5 15.8   15.0 3.0 10 >200B111.4 1.0 0.4 5.0 1.5 3.0  77.0 3.0 1.0 72.0 GROUP V F111.2 1.0 2.3 2.31000 23.0   19.0 2.1 163 >10000 G61.3 1.0 3.5 0.2 5250 16.0   20.0 8.0165 >10000 C42.2 1.0 5.6 16.0 >1000  46.0   76.0 12.0 126 >1000 A71.11.0 2.4 0.5 >100  56.0   11.0 9.0 >100 >100 ^(a)IC₅₀ concentrationrequired for 50% inhibition. ^(b)Underlined valued indicate sites ofspecificity.

TABLE 4 Fine specificity and binding to D-Lys⁸-Cs-BSA and Thr-Cs-BSAconjugates of 22 monoclonal antibodies belonging to groups I-V. Relativebinding to Amino acid residues recognized^(a) BSA-D-Lys⁸-Cs/ Group McAb1 2 3 4 5 6 7 8 9 10 11 BSA-Thr-Cs^(b) I G2 2.1 − − + + − + − − + + + +− − <0.003 I B4 1.2 − − + + − − + − + + + + − − <0.003 I A11 1.1 + − + +− + + − + + + + − − <0.01 I H1 2.4 − − + + − + + − + + + + − − <0.003 ID11 1.3 + − + + − + + − + + + + − − <0.1 I G7 1.1 + − + + − + + − + + +− − <0.02 I C9 1.1 − − + + − − + − + + + + − − <0.03 I H2 1.2 − − + +− + + − + + + + − + <0.003 II D5 1.4 + + + + + + + + + + − − − − − >50II H4 1.3 + + + + + + + + + + + + − − − − − >50 II D31.3 + + + + + + + + + + + − − − − − >50 III B9 1.2 + + − + + + + + + + +− − + − − 20 III H6 1.5 + + + + + + + − + + − − − − − 3 III C4 2.4− + + + + + − + + − − − − − 5 IV D10 1.1 − − − − − − − − + + + − 2 IVA11 2.10 + − − − − − − + + + + + − 2 IV B2 2.4 − − − − − − − − + + + − 2IV B11 1.4 + − − − − − − − + + + + − 2 V C4 2.2 − − + + − − + +− + + + + + + − <0.02 V A7 1.1 − − + + − − + + − + + + + + + + <0.001 VF11 1.2 − − + + − − + + − + + + + + + − <0.06 V G6 1.3 − − + + − − + +− + + + + + + − <0.02 ^(a)+ +, + and − correspond to residues strongly,weakly and not recognized by the McAbs as defined in FIG. 6 and Table 3.^(b)The results are expressed as the ratio between the concentrations ofMcAb required to achieve an identical ELISA response (1.5 Absorbance)when incubated on microtiter plates coated with 0.5 μg/ml D-Lys⁸-Cs-BSAor with Thr²Cs-BSA.

TABLE 5 Recognition of Cs-derivatives modified singly at each amino acidresidue by 5 monoclonal antibodies belonging to the 5 specificitygroups. Replacement McAb G7 1.1 H4 1.3 H6 1.5 B11 1.4 C4 2.2 Originalresidue group I II III IV V MeBint¹ 3′desoxy 0.6 1.2 0.0 0.4 <0.03′-O-acetyl 0.1 1.1 0.6 0.7 0.2 ethylmethyl-Me-Thr <0.0 0.7 <0.0 <0.00.2 8′-hydroxy 0.0 <0.0 <0.0 <0.0 0.0 Abu² aThr <0.0 1.5 1.1 0.0 0.3 Ser<0.0 0.9 0.6 0.0 0.2 Ala 0.2 ND 0.0 0.4 0.1 Sar³ D-MeAla 1.2 1.3 1.4<0.0 1.2 O-Acetyl-MeSer 1.2 2.1 2.2 0.4 1.5 MeLeu⁴ MeAla <0.0 1.8 1.20.3 0.1 MePhe <0.0 0.7 2.3 0.2 0.0 Val⁵ NVa², Nva⁵ 0.5 1.2 0.2 0.2 0.1MeLeu 1.0 1.2 0.3 0.0 <0.0 Met <0.0 1.5 <0.0 0.0 <0.0 MeLeu⁶ McAla 0.70.9 2.7 0.0 1.2 MePhe 0.1 1.2 1.7 0.0 1.3 Ala⁷ Abu <0.0 0.0 0.0 0.0 <0.0D-Ala⁸ D-Ser 1.3 0.0 <0.0 0.1 0.3 D-Thr >2.0 0.0 <0.0 0.4 1.2 Boc-D-Lys2.0 0.6 0.0 0.3 1.2 MeLeu⁹ MeAla 1.4 <0.0 0.0 1.7 1.9 MePhe 0.3 0.0 0.11.2 1.8 MeAla¹⁰ MeAla <0.0 0.4 0.0 0.1 0.8 MePhe <0.0 0.5 0.2 1.6 1.3MeVal¹¹ MeIle 0.4 0.0 0.0 0.2 0.3 aMeIle 0.0 0.3 0.3 <0.0 0.0 Resultsare expressed as the log₁₀ difference in the concentrations ofCs-derivatives and unmodified Cs required to achieve 50% inhibition inELISA. Superscript is position of residue in Cs.

EXAMPLE IV

A. Western Blot to Determine binding to gag Protein

A western blot test was performed according to the procedure outlined inLuban, J. and Goff, S. (1991) Binding of Human Immunodeficiency VirusType 1 (HIV-1) RNA to Recombinant HIV-1 gag Polyprotein. J. Virology65:3203-3212.

Results are demonstrated in FIG. 9. Lane 1 shows the results of the testusing an antibody raised to p24 segment of gag protein. This segment hasthe active sequence in it. Lanes 2 through 23 show the results of thetest using various monoclonal antibodies to cyclosporine A. Lane 9represents the antibody B-11 1.4 Lane 24 is the control with noantibody.

B. Inhibition of binding of B-11 1.4 to p24 by Cyclophilin A

Blocking antibody binding to p24 by cyclophilin A by preincubating thep24-coated plate with (1 ug/ml in same buffer as other ELISA) withpurified human cyclophilin A (a generous gift from Dr. Robert E.Handschumacher, Yale University) at the indicated concentrations in PBSfor 1 hr at 37° C. We observed a linear inhibition of antibody bindingby cyclophilin A indicating that either cyclophilin competed with theantibody for it's binding site or that cyclophilin binding to p24sterically blocked the antibody binding site. See FIG. 10.

C. Inhibition of binding of B-11 1.4 by Cyclosporine A

To demonstrate that the B-11 1.4 monoclonal antibody reacts withnon-denatured p24 we set up an ELISA.

Ninety-six well plates (Corning 25855) were coated with recombinant p24produced in a baculovirus expression system (American Biotechnologies,Inc., Cambridge, Mass.) at 1 ug/ml in 0.1 N NaHCO₃, pH 9.3, for 2.5 hrsat 37° C. Plates were then blocked with 2% fetal calf serum in phosphatebuffered saline with 0.1% Tween-20 for 1 hr at 37° C. B-11 1.4 ascitesfluid was preincubated with Cyclosporine A (Sandoz Research Institute)at the indicated concentrations for 1 hr at 37° C., and then incubatedwith the p24-coated plates for 90 min at 37° C. Bound B-11 1.4 was thendetected in a peroxidase reaction as previously described in Cacalano,et al. Molec. Immunol. 2:107-18 (1992). Specificity of B-11 1.4 bindingto native p24 was demonstrated by the linear inhibition of binding bycyclosporine A. See FIG. 11.

EXAMPLE V

Clinical uses of the compositions of the subject invention

A. Occupational Exposure to HIV-1

Health care workers exposed to HIV-1-contaminated blood or other bodilyfluids can become infected with the virus. Common routes of exposureinclude, but are in no way limited to, the following: penetration of theskin by an uncapped syringe needle coated with HIV-1-containing bodilyfluids (“needle-stick-injury”); cuts caused by scalpels or otherinstruments during surgery on HIV-1-infected individuals; and splashesof blood or other bodily fluids in the eyes or on cracked skin.

To reduce the risk of HIV-l transmission in the health care setting, thecomposition of matter or antibody of the subject invention would beadministered to a health care worker who was exposed toHIV-1-contaminated fluids, by routes such as those listed above. Thesemay be administered by, inter alia, intravenous bolus, continual IVinfusion, intramuscular injection, subcutaneous injection or directly tothe wound or exposed skin. A combination of these routes may be used.Depending on the route of administration and the nature of thetreatment, the composition of the subject invention might be givencontinuously or intermittently. The treatment may be most effective ifthe composition were administered soon after the exposure as possible,for example within one or two hours after exposure.

B. Mother To Infant Transmission of HIV-1

Newborns of HIV-1-infected mothers often become infected with HIV-1. Inmany cases, infection occurs around the time of birth, due to theexposure to the baby to HIV-1-contaminated blood and other bodily fluidsfrom the mother.

To reduce the risk of HIV-1 transmission in this setting, thecomposition of matter or antibody of the subject invention would beadministered to the other prior to delivery, or to the baby afterdelivery, or to both. The possible routes of administration includethose listed surra. The purpose of treating the mother would be toreduce the infectivity of the maternal blood or other bodily fluidsprior to delivery. As an example, the treatment may comprise deliveringto the mother a series of intravenous bolus injections of thecomposition starting several hours or more before birth. Subsequently,the newborn would be treated with the composition in order to reduce theinfectivity of any virus which had entered its body around the time ofbirth. For example, within one or two hours after birth, the newborn maybe treated with a continuous IV infusion of the composition for severaldays.

References

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What is claimed is:
 1. A method of testing whether a composition ofmatter inhibits binding between an HIV-1 p24 gag protein and adetectably labeled cyclosporine-specific antibody which cross reactswith HIV-1 p24 gag protein which comprises: a) immobilizing thecomposition of matter, wherein the composition of matter has thestructure:

wherein a plurality of R's are X and the remainder are H, wherein X is aligand comprising a reactive group and wherein X is bonded to thecomposition of matter by a photochemical reaction between a hydrogen ofcyclosporine A and a photochemically activatable precursor of X; b)contacting the immobilized composition of matter from (a) with a mixtureof HIV-1 p24 gag protein and a detectably labeled antibody whichspecifically binds to N-methyl leucine residues 9 and 10 of cyclosporineand cross reacts with HIV-1 p24 gag protein under conditions allowingfor the labelled antibody to bind to the immobilized composition ofmatter from (a) and form a complex therewith; c) separating any unboundlabeled antibody from any complex formed in (b); d) detecting anylabeled antibody bound to the complex in (b); and e) quantitating theamount of labeled antibody from (d), a decrease in the amount ofantibody bound to the composition of matter after incubation with theHIV-1 p24 gag protein in step (b) compared to the amount of antibodybound to the composition of matter in the absence of HIV-1 p24 gagprotein indicating that the composition of matter inhibits bindingbetween the HIV-1 p24 gag protein and the detectably labeled antibody.2. The method of claim 1 wherein the detectably labeled antibody whichspecifically binds to N-methyl leucine residues 9 and 10 of cyclosporineis the monoclonal antibody B-11 1.4 produced by the hybridoma cell linehaving ATCC Accession No. HB-11853.
 3. The method of claim 2 wherein thedetectably labeled antibody is labeled with an enzyme, dye, fluorescentmarker, colored bead, radioactive isotope or biotin.
 4. A kit fortesting whether a composition of matter inhibits binding between HIV-1p24 gag protein and a detectably labeled cyclosporine-specific antibodywhich cross reacts with HIV-1 p24 gag protein which comprises: a) aplate comprising a plurality of wells; b) the composition of matterimmobilized upon the wells, wherein the composition of matter has thestructure:

wherein a plurality of R's are X and the remainder are H, wherein X is aligand comprising a reactive group and wherein X is bonded to thecomposition of matter by a photochemical reaction between a hydrogen ofcyclosporine A and a photochemically activatable precursor of X; and c)a solution comprising the detectably labeled cyclosporine-specificantibody which cross reacts with HIV-1 p24 gag protein and whichspecifically binds to N-methyl leucine residues 9 and 10 ofcyclosporine; and d) a solution comprising the HIV-1 p24 gag protein. 5.The kit of claim 4 wherein the detectably labeled antibody whichspecifically binds to N-methyl leucine residues 9 and 10 of cyclosporineis the monoclonal antibody B-11 1.4 produced by the hybridoma cell linehaving ATCC Accession No. HB-11853.
 6. The kit of claim 4 wherein thedetectably labeled antibody is labeled with an enzyme, dye, fluorescentmarker, colored bead, radioactive isotope or biotin.